Rhizobia are Gram-negative soil bacteria that form nitrogen-fixing symbioses with legumes. A specific rhizobial species recognizes and infects a specific host plant. Rhizobial cell surface lipopolysaccharides (LPSs) are important for endocytotic invasion of host root nodule cells and differentiation into nitrogen-fixing bacteroids within a host-derived intracellular compartment known as the symbiosome. The symbiosome membrane is derived from the Golgi and endoplasmic reticulum of the host cell and is both acidic and low in O2. Rhizobial symbionts are analogous to animal bacterial pathogens that survive in phagosome-derived intracellular compartments and cause chronic infections; e.g. Brucella. The LPSs from members of the Rhizobiaceae have a very long chain lipid-A fatty acid, 27-OHC28:0, (as does the pathogen, Brucella abortus). Also, LPSs from R. etli (Re) (a bean symbiont) and from R. leguminosarum bv. vicae (Rlv) (a pea symbiont) have unique lipid-A, and core oligosaccharide; e.g. they are devoid of phosphate and contain galacturonic acid. During symbiosis, modifications are made to the O-chain polysaccharide (e.g. methylation) and lipid-A. In the case of Rlv, both LPS and the entire bacterium become hydrophobic. This increase in hydrophobicity is due primarily to a doubling of the lipid-A 27-OHC28:0 moiety. It is proposed that the LPS O-chain modifications are required for adherence of the bacterium to the host membrane, and that the 27-OHC28:0 moiety is required to maintain membrane stability during endocytosis and symbiosome formation. The aims of this proposal are to structurally characterize the O-chain modifications, and to determine the functions of the Re and Rlv unique structural features with regard to both symbiosis and LPS biosynthesis. This will be accomplished through the creation and analysis of Re and Rlv mutants that are specifically altered in these unique structural features (e.g. defective in 27-OHC28:0 incorporation), and by the isolation and characterization of LPS fragments that bind to specific monoclonal antibodies whose LPS epitopes change during symbiosis.